Bacillus anthracis, Francisella tularensis and Yersinia pestis, the causative agents for anthrax, tularemia and plague, respectively, are all classed as Category A agents due to their potential use in bioterrorism and biowarfare. Literature clearly demonstrates that multi-drug resistant strains for all three have either been isolated from natural sources, or can be readily selected through standard selection processes. Further, although the wild-type strains of all three are variously treatable with current antibiotics, none of these antibiotics is ideal for treatment of these diseases, and only doxycycline is generally indicated for more than one agent (anthrax and plague). Thus, there is a clear imperative for developing new therapeutic agents against all three organisms. In preliminary work, we have developed inhibitors of the enzyme enoyl reductase, or FabI, that show strong efficacy against both B. anthracis and F. tularensis, as well as a variety of other pathogens. Additionally, MenE, an essential enzyme in menaquinone biosynthesis is predicted to to be essential for all three organisms, and has properties well suited for development as an antibiotic target for all three organisms. Based on extensive preliminary studies with both enzymatic systems, we propose to discover and biologically evaluate lead inhibitors of both FabI and MenE in this two-year ARRA-funded project. We will utilize an iterative process of structure-based molecular design, in silico screening of large compound libraries, high throughput screening of selected compounds, synthetic medicinal chemistry, initial in vitro toxicology evaluation, and initial animal proof of principle studies to discover compounds with potential efficacy against the three Category A pathogens, B. anthracis, F. tularensis and Y. pestis. The two targets in different metabolic pathways will provide advantageous complementary potential for lead and back-up compounds with very different characteristics. The goal of this U01 application is to develop an advanced series of broad spectrum antibacterial lead compounds that are safe, efficacious, and orally bioavailable in established animal models. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page 85 Continuation Format Page